Bone graft materials and methods

ABSTRACT

Compositions, materials, methods and kits for bone grafting are described. In some embodiments, a bone graft composition includes about 15% to about 20% by weight collagen, about 55% to about 70% by weight bioactive glass, and about 15% to about 30% by weight a calcium phosphate. The bioactive glass and the calcium phosphate together are about 80% to about 85% by weight of the bone graft composition. In some embodiments, a bone graft composition includes a collagen matrix and a plurality of bioactive glass particulates dispersed throughout the collagen matrix. The collagen matrix is about 20% to about 60% by weight of the bone graft composition, and the bioactive glass is about 40% to about 80% of the bone graft composition. In some embodiments, a majority of the bioactive glass particulates are about 53 μm to about 425 μm in size.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/047,677, filed Oct. 7, 2013, entitled “Bone Graft Materials andMethods,” which is a continuation of and claims priority to U.S. patentapplication Ser. No. 12/977,191, filed Dec. 23, 2010, entitled “BoneGraft Materials and Methods,” the disclosures of which are incorporatedby reference herein in their entireties.

BACKGROUND

The present invention generally relates to bone grafting, and moreparticularly to repairing and/or filling a void or gap in a bone or bonystructure of a patient.

A need exists for improved bone graft materials. Current bone graftingincludes the use of autogenous bone as a graft material (i.e.,“autografting”). Use of autogenous bone, however, subjects a patient toincreased pain and discomfort, and an increased risk of infection,because it requires the patient undergo surgery to recover theautogenous bone for use in the grafting procedure. Current bone graftingalso includes the use of bone from a donor as a graft material (e.g.,“allografting” from the same species or “xenografting” from a differentspecies). Both allograft bone and xenograft bone, though from naturalsources, subject a patient to the risk of disease transmission and graftrejection.

Current bone grafting further includes the use of synthetic bone graftmaterial. Some such synthetic bone graft material is mixed withautograft, allograft, or xenograft bone, and thus still subjects apatient to the risks above. Other disadvantages to current syntheticbone graft material are the lack of sufficient resorbability, lack ofsufficient porosity, and increased manufacturing costs due to a highnumber of component materials. As such, there is a need for an improvedsynthetic bone graft material that is resorbable and porous, and thathelps to reduce manufacturing costs by reducing the number of componentmaterials.

SUMMARY OF THE INVENTION

Compositions, materials, methods and kits for bone grafting aredescribed. In some embodiments, a bone graft composition includes about15% to about 20% by weight collagen, about 55% to about 70% by weightbioactive glass, and about 15% to about 30% by weight a calciumphosphate. The bioactive glass and the calcium phosphate together areabout 80% to about 85% by weight of the bone graft composition. In someembodiments, a bone graft composition includes a collagen matrix and aplurality of bioactive glass particulates dispersed throughout thecollagen matrix. A majority of the bioactive glass particulates areabout 53 μm to about 425 μm in size. The collagen matrix is about 20% toabout 60% by weight of the bone graft composition, and the bioactiveglass is about 40% to about 80% by weight of the bone graft composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a posterior view of a bone graft composition according to anembodiment implanted between transverse processes of vertebra.

FIG. 1B is a side view of bone graft compositions according toembodiments disposed between vertebral bodies and on posterior portionsof vertebrae.

FIG. 1C is a side view of a bone graft composition according to anembodiment disposed proximate to a facet joint of a spine.

FIG. 1D is an anterior view of a bone graft composition according to anembodiment disposed on an ilium.

FIG. 1E is an anterior view of bone graft compositions according toembodiments disposed at an iliac crest and an acetabulum.

FIG. 1F is a side view of a bone graft composition according to anembodiment disposed in a radius (which is shown adjacent an ulna).

FIG. 1G is a perspective view of a bone graft composition according toan embodiment disposed in a femur.

FIG. 1H is a side view of bone graft compositions according toembodiments disposed on bones of a foot and at an ankle joint.

FIG. 2A is a perspective view of a bone graft material according to anembodiment.

FIG. 2B is the bone graft material of FIG. 2A implanted into a bone voidin a cranium.

FIGS. 3-6 are top views of bone graft materials according toembodiments.

FIGS. 7-9 are perspective views of bone graft materials according toembodiments.

FIG. 10 is a flow chart of a method of making a bone graft materialaccording to an embodiment.

FIGS. 11A-11C are representative images of a sample preparation of abone graft composition according to Example 3 at day 2, day 7, and day11, respectively.

FIGS. 12A-12C are stained representative images of the samplepreparation of a bone graft composition according to Example 3 at day11.

FIG. 13 is a chart comparing cell confluence of sample preparations ofbone graft compositions according to Examples 3, 4, and 5.

FIG. 14 is a chart comparing mineralization levels of samplepreparations of bone graft compositions according to Examples 3, 4, and5.

FIGS. 15A-15C are representative images of a sample preparation of abone graft composition according to Example 4 at day 2, day 7, and day11, respectively.

FIGS. 16A-16C are stained representative images of the samplepreparation of a bone graft composition according to Example 4 at day11.

FIGS. 17A-17C are representative images of a sample preparation of abone graft composition according to Example 5 at day 2, day 7, and day11, respectively.

FIGS. 18A-18C are stained representative images of the samplepreparation of a bone graft composition according to Example 3 at day11.

FIG. 19 is a chart comparing cell confluence of sample preparations ofbone graft compositions according to Examples 6, 7, and 8.

FIG. 20 is a chart comparing mineralization levels of samplepreparations of bone graft compositions according to Examples 6, 7, and8.

FIGS. 21A-21C are representative images of a sample preparation of abone graft composition according to Example 6 at day 2, day 7, and day11, respectively.

FIGS. 22A-22C are stained representative images of the samplepreparation of a bone graft composition according to Example 6 at day11.

FIGS. 23A-23C are representative images of a sample preparation of abone graft composition according to Example 7 at day 2, day 7, and day11, respectively.

FIGS. 24A-24D are stained representative images of the samplepreparation of a bone graft composition according to Example 7 at day11.

FIGS. 25A-25C are representative images of a sample preparation of abone graft composition according to Example 8 at day 2, day 7, and day11, respectively.

FIGS. 26A-26C are stained representative images of the samplepreparation of a bone graft composition according to Example 8 at day11.

DETAILED DESCRIPTION

Compositions, materials, methods and kits for bone grafting, includingfor repairing and/or filling a void or gap in a bone or other bonystructure of a patient, are described herein. Also described herein aremethods for preparing such compositions and materials.

As used in this specification, the singular forms “a,” “an” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, the term “a material” is intended to mean one or morematerials, or a combination thereof.

As used herein, the term “biocompatible” refers to the ability (e.g., ofa composition or material) to perform with an appropriate host responsein a specific application, or at least to perform without having a toxicor otherwise deleterious effect on a biological system of the host,locally or systemically.

As used herein, the term “osteoconductive” refers to the ability (e.g.,of a composition or material) to passively permit bone growth (e.g.,onto and/or into the material). As such, osteoconduction can becharacterized as a passive process. A material (e.g., a graft orimplant) can be osteoconductive, for example, because it is configuredto passively permit growth of bone on a surface of the material. Inanother example, a material can be osteoconductive because it isconfigured to passively permit growth of bone into an opening (e.g., apore) of the material.

As used herein, the term “osteoinductive” refers to the capability(e.g., of a composition or material) to actively stimulate a biologicalresponse which induces bone formation. As such, osteoinduction can becharacterized as an active process. Osteoinduction can include theformation and/or stimulation of osteoprogenitor cells, such asosteoprogenitor cells in bodily tissue surrounding or proximate to agraft or implant.

As used herein, the term “biodegradable” refers to the capability of amaterial to be degraded, disassembled, and/or digested over time byaction of a biological environment (including the action of livingorganisms, e.g., the patient's body) and/or in response to a change inphysiological pH or temperature. As used herein, the term “resorbable”refers to the capability of a material to be broken down over a periodof time and assimilated into the biological environment.

As used herein, references to a weight of components of a bone graftcomposition or material described herein, such as the phrase “byweight,” refer to the weight of the applicable component prior to beingadded to or mixed with another different component of the bone graftcomposition. For example, the weight can refer to an initial weight ofthe component measured out before further processing of the componentinto the bone graft composition.

As used herein, the term “fibrillar” refers to being in the form offibrils, and not in the form of fibers. For example, a reference tocollagen in the fibrillar form includes collagen fibrils, but not nativecollagen fibers.

As used herein, the phrase “non-load bearing application” refers to anapplication for repair of a void or gap in a bone or another bonystructure in which the void or gap to be repaired is not intrinsic tothe stability of the bone or bony structure.

A bone graft composition (or material) according to an embodiment isconfigured to facilitate repair or regeneration of bone at a targetrepair site. For example, in some embodiments, the bone graftcomposition can be osteoconductive, osteoinductive, or both. The targetrepair site can be, for example, a void, gap, or other defect in a boneor other bony structure in a body of a patient. For example, asdescribed in more detail below, the bone graft composition can beconfigured to facilitate bone growth at a target repair site in thespine, pelvis, an extremity, the cranium, or another bone or bonystructure in the patient's body. The bone graft composition isconfigured to be implanted or otherwise disposed at the target repairsite. For example, in some embodiments, the bone graft composition isconfigured to be implanted or disposed at the target repair site in anon-load bearing application.

The bone graft composition can include various combinations of collagen,bioactive glass, and calcium phosphate, each of which components isdescribed in more detail herein. The bone graft composition isbiocompatible. The bone graft composition is biodegradable. Morespecifically, in some embodiments, at least a portion of the bone graftcomposition is resorbable. For example, at least one of the collagen,bioactive glass, and calcium phosphate, or a combination thereof, can beresorbable. In some embodiments, the bone graft composition is, as awhole, resorbable.

The collagen can be or include soluble collagen, insoluble collagen, ora combination thereof. The collagen can be or include type I collagen,type II collagen, type III collagen, type VII collagen, another suitabletype of collagen, or a combination thereof. For example, in someembodiments, the collagen is or includes medical grade type I collagen.In some embodiments, the collagen includes type I collagen and up toabout 5% of a type of collagen different than type I collagen. Forexample, the collagen can include type I collagen and up to about 5%type III collagen. Specifically, the collagen can include about 5% typeIII collagen and the remainder of the collagen is type I collagen. Inanother example, the collagen can include type I collagen and up toabout 5% type VII collagen. The collagen can be human, equine, bovine,porcine, murine, synthetic, or from another suitable source. Forexample, the collagen can be derived from bovine corneum.

In some embodiments, the collagen is in fibrillar form. In someembodiments, at least prior to being implanted into the body of thepatient, the collagen is not mineralized. In some embodiments, thecollagen is uncompressed. In this manner, because the collagen isuncompressed, the bone graft composition can also be characterized asbeing uncompressed.

The collagen of the bone graft composition can be a matrix in and/or onwhich the bioactive glass and calcium phosphate are disposed. In thismanner, the collagen matrix facilitates delivery of the bioactive glassand calcium phosphate to the target repair site. The collagen matrix ofthe bone graft composition can be in any suitable form. For example, insome embodiments, the collagen matrix is in a flowable form. Suitableflowable forms include a slurry, foam, gel, or paste. In this manner, atleast one of the bioactive glass and/or calcium phosphate can be mixedwith and/or embedded into the flowable collagen matrix. In someembodiments, the collagen matrix is a hardened, brittle, or otherwisedry cracker-like material. For example, the collagen matrix can beformed by drying the flowable collagen, as described in more detailbelow. At least a portion of the bioactive glass and/or the calciumphosphate can disposed (e.g., sprinkled or otherwise coated) onto asurface of the dried collagen matrix. In some embodiments, the collagenmatrix is in a sponge-like form. For example, the dried collagen matrixcan be wetted with a suitable solution to form a sponge-like collagenmatrix. Suitable solutions include, but are not limited to, blood,marrow, another bodily fluid, a simulated body fluid, saline, phosphatebuffered saline, gel, or another biocompatible fluid, or any combinationof the foregoing. In some embodiments, the dried collagen can be wettedwith a solution that includes at least one of the bioactive glass orcalcium phosphate. The collagen matrix, in any suitable form generallyand in the dry or sponge-like form particularly, includes a surfaceconfigured to receive bioactive glass and/or calcium phosphate, forexample, in granular or particulate form.

The collagen matrix of the bone graft composition is porous. In someembodiments, the collagen matrix defines a plurality of pores (e.g., asshown in FIGS. 2A-2B with respect to an implant 100). At least a portionof the pores can be configured to permit the in-growth of bone. In thismanner, the collagen matrix, and thus the bone graft composition, isosteoconductive. The porosity of the collagen matrix can be in anysuitable range. For example, in some embodiments, the bone graftcomposition has a porosity within the range of about 50% to about 95%.In some embodiments, the bone graft composition has a porosity withinthe range of about 70% to about 90%. More specifically, in someembodiments, the bone graft composition is about 70% porous.

The pores of the collagen matrix can be any suitable size(s) forpermitting bone growth therein. For example, in some embodiments, thepores of the collagen matrix each have a diameter greater than about 100μm. In other embodiments, the collagen matrix defines pores each havinga diameter less than about 100 μm. The collagen matrix can define poresof various sizes. For example, in some embodiments, a first portion ofpores of the plurality each have a diameter greater than about 100 μmand a second portion of pores of the plurality each have a diameter lessthan about 100 μm. In some embodiments, at least a portion of theplurality of pores of the composition are interconnected, which canfurther facilitate the in-growth of bone.

The bioactive glass of the bone graft composition is configured tofacilitate the regrowth of bone at the target repair site. In someembodiments, the bioactive glass of the bone graft composition can be anosteoconductive agent. As described above, the bioactive glass can bedisposed on, embedded within, and or mixed with the collagen of the bonegraft material. In some embodiments, the bioactive glass can be mixedwith the collagen such that the bioactive glass is randomly dispersedthroughout the collagen. For example, the bioactive glass can be mixedwith the collagen to form a substantially homogenous mixture (e.g., aslurry) of collagen and bioactive glass. In some embodiments, thebioactive glass is disposed on (e.g., coated or sprinkled onto) asurface of the collagen (e.g., the collagen matrix in one of theflowable, dried, or sponge-like forms).

The bioactive glass can be any alkali-containing ceramic, glass,glass-ceramic, or crystalline material that facilitates bone formationafter contact with a biological environment. Suitable bioactive glasscan include 45S5, 58S, S70C30, or a combination of the foregoingbioactive glasses. Specifically, in some embodiments, the bioactiveglass is a 45S5 Bioglass with a nominal chemical composition of 45%silicon dioxide (SiO₂) (±2%), 24.5% calcium oxide (CaO) (±2%), 24.5%sodium oxide (Na₂O) (±2%), and 6% phosphorous pentoxide (P₂O₅) (±1%).The bioactive glass can include trace or minimal amounts of at least oneheavy element, including, but not limited to, arsenic (As), cadmium(Cd), mercury (Hg), lead (Pb), or a combination thereof. For example,the bioactive glass can include As in an amount less than about 3 partsper million (ppm). In another example, the bioactive glass can includeCd in an amount less than about 5 ppm. In yet another example, thebioactive glass can include Hg in an amount less than about 5 ppm. Instill another example, the bioactive glass can include Pb in an amountless than about 30 ppm. Specifically, in some embodiments, the bioactiveglass is a 45S5 Bioglass of the composition described above andincluding 3 ppm As, 5 ppm Cd, 5 ppm Hg, and 30 ppm Pb.

The bioactive glass can be in any suitable form. For example, in someembodiments, the bioactive glass is in particulate form. In theparticulate form, the bioactive glass particles are discrete andgenerally not interconnected. As such, the bioactive glass particles,collectively, are generally amorphous. In other words, the bioactiveglass particles, collectively, generally lack an intentional structureor organization. The bioactive glass particles can be generallyirregular in shape. The bioactive glass particles can have a smoothsurface.

The bioactive glass particles can be any suitable size. In someembodiments, at least a portion of the bioactive glass particles arewithin a range of about 53 μm to about 425 μm. In some embodiments, thebioactive glass includes particles within a range of about 212 μm toabout 425 μm. For example, in some embodiments, at least 85% of thebioactive glass are particles within a range of about 212 μm to about425 μm. The bioactive glass can include particles of various sizes; forexample, of various sizes within at least one of the foregoing ranges.In some embodiments, the bioactive glass particles are sufficientlylarge to prevent the particles from leaching out of the collagencarrier, e.g., when the dried collagen is wetted with a solution.

Any suitable method of measuring the bioactive glass particle size maybe used. For example, the bioactive glass particles can be sieved usingASTM sieves according to ASTM E 11-70 (1995) method. When using such amethod, for example, particles (or granules) retained between 40 and 70mesh can be used in the bone graft composition. Because particlesscreened within a certain range may contain a small amount of smallerparticles due to screen blinding, a precision screen may be used todetermine the amount of particles within the desired particle sizerange.

The calcium phosphate of the bone graft composition is also configuredto facilitate the regrowth of bone at the target repair site. In someembodiments, the calcium phosphate of the bone graft composition is anosteoinductive agent. The calcium phosphate is configured to be disposedon, embedded in, or otherwise mixed with the collagen. In someembodiments, the calcium phosphate can be mixed with the collagen suchthat the calcium phosphate is randomly dispersed throughout thecollagen. For example, the calcium phosphate can be mixed with thecollagen to form a substantially homogenous mixture (e.g., a slurry) ofcollagen and calcium phosphate. In another example, the calciumphosphate can be mixed with the collagen and the bioactive glass.

The calcium phosphate can include any suitable calcium phosphate ormineral thereof, including, but not limited to, hydroxyapatite(sometimes referred to as hydroxylapatite; also referred to herein as“HA”), tricalcium phosphate (also referred to herein as “TCP”), or acombination of the foregoing. In some embodiments, the calcium phosphateis biphasic and includes tricalcium phosphate and hydroxyapatite. Forexample, the calcium phosphate can include about 40% to about 80% byweight tricalcium phosphate and about 20% to about 60% by weighthydroxyapatite. More specifically, in some embodiments, the calciumphosphate includes about 80% tricalcium phosphate and about 20%hydroxyapatite. In other embodiments, the calcium phosphate includesabout 60% tricalcium phosphate and about 40% hydroxyapatite. In yetother embodiments, the calcium phosphate includes about 40% tricalciumphosphate and about 60% hydroxyapatite.

The calcium phosphate can be in any suitable form. For example, thecalcium phosphate can be in particulate or granular form. The calciumphosphate can be of any suitable size. For example, in some embodiments,the calcium phosphate includes mineral particles within the range ofabout 200 μm to about 2 mm in size. In some embodiments, the calciumphosphate includes mineral particles within the range of about 200 μm toabout 800 μm in size. In some embodiments, the calcium phosphateincludes mineral particles within the range of about 0.5 mm to about 1mm in size.

Bone graft compositions of various weight ratios of collagen, bioactiveglass, and calcium phosphate are contemplated. In some embodiments, abone graft composition includes about 10% to about 20% by weightcollagen, about 25% to about 80% bioactive glass, and about 5% to about60% calcium phosphate. More specifically, a bone graft compositionaccording to an embodiment includes about 15% to about 20% by weightcollagen, about 55% to about 70% by weight bioactive glass, and about15% to about 30% by weight calcium phosphate. The bioactive glass andthe calcium phosphate together comprise about 80% to about 85% by weightof the bone graft composition.

In some embodiments, for example, the bone graft composition can includeabout 15% by weight collagen, about 55% to about 65% by weight bioactiveglass, and about 20% to about 30% by weight calcium phosphate. Inanother example, the bone graft composition can include about 15%collagen, about 55% bioactive glass, and about 30% calcium phosphate,such that a weight ratio of the collagen to the bioactive glass to thecalcium phosphate is about 15%:55%:30%, respectively.

In yet another example, the bone graft composition can include about 15%collagen, about 60% bioactive glass, and about 25% calcium phosphate,such that a weight ratio of the collagen to the bioactive glass to thecalcium phosphate is about 15%:60%:25%, respectively. In still anotherexample, the bone graft composition can include about 15% collagen,about 65% bioactive glass, and about 20% calcium phosphate, such that aweight ratio of the collagen to the bioactive glass to the calciumphosphate is about 15%:65%:20%, respectively.

In other embodiments, the bone graft composition can include about 20%by weight collagen, about 50% to about 60% by weight bioactive glass,and about 20% to about 30% by weight calcium phosphate. For example, thebone graft composition can include about 20% collagen, about 50%bioactive glass, and about 30% calcium phosphate, such that a weightratio of the collagen to the bioactive glass to the calcium phosphate isabout 20%:50%:30%, respectively.

In another example, the bone graft composition can include about 20%collagen, about 55% bioactive glass, and about 25% calcium phosphate,such that a weight ratio of the collagen to the bioactive glass to thecalcium phosphate is about 20%:55%:25%, respectively. In still anotherexample, the bone graft composition can include about 20% collagen,about 60% bioactive glass, and about 20% calcium phosphate, such that aweight ratio of the collagen to the bioactive glass to the calciumphosphate is about 20%:60%:20%, respectively. In some embodiments, thecollagen, bioactive glass, and calcium phosphate collectively comprise100% by weight of the bone graft composition.

In the foregoing examples, the collagen, bioactive glass, and calciumphosphate can be any collagen, bioactive glass, and calcium phosphate,respectively, described herein. For example, the collagen can be medicalgrade type I collagen. In another example, the calcium phosphate caninclude about 40% to about 80% tricalcium phosphate and about 20% toabout 60% hydroxyapatite.

Although the bone graft compositions have been described above asincluding collagen, bioactive glass, and calcium phosphate, in someembodiments, a bone graft composition includes collagen and bioactiveglass. For example, a bone graft composition according to anotherembodiment includes a collagen matrix and a plurality of bioactive glassparticulates. The collagen matrix can include any collagen describedherein, or of a combination of any collagen described herein. Forexample, the collagen matrix can be or include type I collagen. Inanother example, the collagen matrix can be or include a combination oftype I collagen and type III collagen. The collagen matrix can be in anysuitable form. For example, in some embodiments, the collagen matrix isin a flowable form (e.g., a slurry, foam, gel, or paste), a dried form,or a sponge-like form, as described above. In some embodiments, theplurality of bioactive glass particulates is dispersed throughout thecollagen matrix. The bioactive glass can be any bioactive glassdescribed herein. For example, in some embodiments, a majority of theplurality of bioactive glass particulates are within a range of about 53μm to about 425 μm in size. More specifically, in some embodiments, themajority of the bioactive glass particulates can be within a range ofabout 212 μm to about 425 μm in size. Still more specifically, in someembodiments, at least 85% of the plurality of bioactive glassparticulates can be within the range of about 212 μm to about 425 μm insize.

Bone graft compositions of various ratios of collagen and bioactiveglass are contemplated. In some embodiments, the bone graft compositionincludes, by weight, about 20% to about 60% collagen matrix and about40% to about 80% bioactive glass. More specifically, for example, thecollagen matrix can be about 20% by weight of the bone graft compositionand the plurality of bioactive glass particulates can be about 80% byweight of the bone graft composition. In another example, the collagenmatrix can be about 40% by weight of the bone graft composition and theplurality of bioactive glass particulates can be about 60% by weight ofthe bone graft composition. In yet another example, the collagen matrixcan be about 60% by weight of the bone graft composition and theplurality of bioactive glass particulates can be about 40% by weight ofthe bone graft composition.

In some embodiments, the collagen matrix and the plurality of bioactiveglass particulates collectively comprise 100% by weight of the bonegraft composition. In embodiments in which the collagen and bioactiveglass comprise 100% of the bone graft composition, the bone graftcomposition is, prior to implantation into the patient's body, free ofadditional components including, but not limited to, bone or formsthereof (e.g., bone particles, bone powder, demineralized bone matrix),cells, tissue particles, blood products, calcium phosphate, rubber,gelatin, bone morphogenetic proteins, growth factors, anti-inflammatoryagents, drugs, and radiopaque particles.

As noted above, a bone graft composition according to an embodiment canbe configured for use at various target repair sites within a body of apatient to facilitate bone growth therein. In some embodiments, the bonegraft composition is configured for use at a target repair site in thepatient's spine. For example, as shown in FIG. 1A, a bone graftcomposition 10 can be disposed in an opening between a transverseprocess of a first vertebra and a transverse process of a secondvertebra. In this manner, the bone graft composition can facilitategrowth of a bony bridge between the transverse processes of the firstand second vertebrae, such as to achieve posterolateral spinal fusion.In another example, as shown in FIG. 1B, a bone graft composition 20 canbe disposed in a void or opening between a body of a first vertebra anda body of a second vertebra different than the first vertebra. In thismanner, for example, the bone graft composition can facilitate growth ofbone between the body of the first vertebra and the body of the secondvertebra to achieve interbody fusion of the vertebrae. Referring againto FIG. 1B, in some embodiments, a plurality of bone graft compositionimplants 22 can be positioned adjacent a posterior portion of the spine,for example, to facilitate growth of a bony bridge between adjacentvertebrae. In this manner, the plurality of bone graft compositionimplants 22 can facilitate fusion of the adjacent vertebrae. In someembodiments, such as in a spinal fusion procedure, the bone graftcomposition is used in conjunction with a mechanical support (e.g., aplurality of screws and/or rods, as shown in FIG. 1B). In still anotherexample, referring to FIG. 1C, a bone graft composition 30 can beimplantable in or proximate to a facet joint of adjacent vertebrae tofacilitate growth of bone at the facet joint.

In some embodiments, a bone graft composition is configured for use at atarget repair site in the patient's pelvis. For example, as shown inFIG. 1D, a bone graft composition 40 can be disposed in an opening inthe patient's ilium. In some embodiments, a bone graft composition isconfigured to be disposed in or at a target repair site at a differentportion of the pelvis, such as, for example, the iliac crest (e.g., bonegraft composition 50 shown in FIG. 1E), acetabulum (e.g., bone graftcomposition 52 shown in FIG. 1E), ischium, or pubis.

In some embodiments, a bone graft composition is configured for use at atarget repair site in a bone of an extremity of the patient. Forexample, a bone graft composition can be configured to be disposed in anopening in the radius (e.g., bone graft composition 60 in FIG. 1F),ulna, humerus, tibia, fibula, femur (e.g., bone graft composition 70 inFIG. 1G), or other bone of an extremity. In another example, the bonegraft composition can be configured to be disposed in an opening in aknee joint. In yet another example, referring to FIG. 1H, a bone graftcomposition is configured to be disposed in an opening in a bone of thepatient's foot. For example, in some embodiments, the bone graftcomposition is configured to be disposed in an opening of a calcaneus(i.e., heel bone) (e.g., bone graft composition 80), navicular (e.g.,bone graft composition 82), talus, cuboid, or cuneiform bone of thefoot. In another example, referring to FIG. 1H, a bone graft compositioncan be in the form of an implant 84 configured to be disposed at atarget repair site in or proximate to an ankle joint, i.e., between thetibia and the talus.

In some embodiments, referring to FIGS. 2A-2B, the bone graftcomposition can be in the form of an implant 100 configured for use inor at a target repair site in a patient's cranium to facilitate growthof bone therein. Although specific examples of suitable target repairsites have been illustrated and described, in other embodiments, thebone graft composition can be configured to be implanted into or at atarget repair site in a different bone or bony structure of thepatient's body.

A bone graft material kit according to an embodiment includes at least acollagen (e.g., a collagen matrix), as described above, and bioactiveglass (e.g., in the form of particles), as described above. In someembodiments, the kit includes calcium phosphate, for example in thecollagen matrix as described above. The bioactive glass of the kit canbe maintained separately within the kit from the collagen. For example,the bioactive glass particles can be disposed in a vial during themanufacturing stage. The vial of bioactive glass particles is packagedwith the collagen matrix for delivery to a patient treatment facility.In some embodiments, the bioactive glass particles are included in asolution contained in the vial. The collagen can be separately sealedwithin the kit. In some embodiments, the collagen matrix is in a secondvial, such as when the collagen matrix is in the form of a slurry orfoam. In some embodiments, the collagen matrix is sealed within foil orother packaging within the kit, such as when the collagen matrix is inthe dried or sponge-like form. In this manner, the bioactive glass canbe added by the physician or other healthcare practitioner to thecollagen in any suitable manner described herein at a desired time priorto implanting the bone graft material at the target repair site.

A method 180 of making a bone graft material according to an embodimentis described herein with reference to the flowchart in FIG. 10. The bonegraft material can include, for example, any bone graft compositiondescribed herein.

In some embodiments, the method optionally includes preparing collagenfor inclusion in the bone graft material. The collagen can include anycollagen described herein, or combination thereof. The preparing caninclude preparing the collagen to be a matrix or carrier configured tobe implanted at a target repair site and to deliver other components(e.g., an osteoconductive agent, an osteoinductive agent, bioactiveglass, calcium phosphate, etc.) to the target repair site. In someembodiments, the preparing the collagen includes preparing collagen thatincludes type I collagen and type III collagen. In a specific example,the prepared collagen can be collagen a mixture of type I collagen andtype III collagen derived from limed bovine corneum where half of thecollagen mixture is base processed and half of the collagen mixture isacid swollen gel. In one embodiment, the preparing collagen includeschilling a desired amount of type I collagen at about 2° to about 10° C.For example, the preparation of type I collagen can be chilled until thetype I collagen weighs about 100 mL at equilibrium. The preparingcollagen includes weighing out a desired amount of type III collagen.For example, an about 1 gram preparation of type III collagen can beweighed out. The preparing collagen further includes mixing the type IIIcollagen with the type I collagen. The mixture of type III collagen andtype I collagen can optionally be stored (e.g., overnight) at about 2°to about 10° C. Although the prepared collagen is described hereinincluding type I collagen and type III collagen, in other embodiments,the prepared collagen includes type I collagen and a type of collagendifferent than type I or type III collagen (e.g., type VII collagen).

Optionally, a phosphate solution is added to the mixture of type IIIcollagen and type I collagen. For example, 0.2 M phosphate with about0.13 M NaCl can be added to the collagen mixture. Specifically, a 10 mLpreparation of the phosphate solution can be added to the collagenmixture. Optionally, the collagen mixture with the added phosphate istitrated. For example, the collagen mixture with the added phosphatesolution can be titrated with 1 N NaOH until the pH of the mixture iswithin the range of about 7.0 to about 7.8. More specifically, themixture can have a pH of 7.4. Optionally, the titrated mixture is mixed.For example, the titrated mixture can be mixed for about one minute.Optionally the mixture is held for a desired period of time at a desiredtemperature with no agitation. For example, the mixture can be left atrest for at least 10 hours at about 2 to about 10° C. with no agitation.

In some embodiments, the method optionally includes weighing out atleast one component to be included in the bone graft material. Forexample, a desired dry weight of at least one component (e.g., thecalcium phosphate and/or the bioactive glass) can be weighted out. Inanother example, in some embodiments, the collagen is in a flowable form(such as a slurry of collagen and water). As such, a desired dry weightof collagen is calculated based on the concentration, e.g., of theslurry, and is weighed out volumetrically. For example, to obtain 2grams of collagen from a slurry having a concentration of 20 mg ofcollagen per 1 mL of liquid, a 100 mL collagen slurry is volumetricallyweighed out. In one embodiment for making a bone graft materialincluding collagen, calcium phosphate, and bioactive glass, the methodincludes weighing out a desired amount of at least one of the collagen,the calcium phosphate, and the bioactive glass. In yet another example,an embodiment for making a bone graft material including collagen andbioactive glass can include weighing out a desired amount of at leastone of the collagen and the bioactive glass.

At step 182, the method 180 includes mixing collagen with calciumphosphate. For example, the mixing can include mixing the weighed outamounts of collagen and calcium phosphate. In some embodiments, themixing includes mixing the collagen into a slurry or foam. The mixing atstep 182 can include pouring the calcium phosphate (e.g., in the form ofgranules) into the collagen. The calcium phosphate is mixed with thecollagen (e.g., using a spatula or other apparatus for hand-mixing thecomponents). The collagen and calcium phosphate can optionally be mixeduntil the mixture is substantially homogenous (e.g., until the mixturevisually appears to be homogenous).

Optionally, the mixing at step 182 includes mixing bioactive glass withthe collagen and calcium phosphate. For example, a weighed out amount ofbioactive glass particles can be poured into and mixed with the collagenin a similar manner as described above with respect to mixing thecalcium phosphate with the collagen. The bioactive glass and calciumphosphate can be added to and mixed with the collagen consecutively(with either of the bioactive glass and the calcium phosphate beingadded before the other of the bioactive glass and the calcium phosphate)or concurrently. The mixture of collagen and calcium phosphate, andoptionally bioactive glass, is referred to herein with respect to method180 as the collagen mixture.

At step 183, the method 180 optionally includes cross-linking thecollagen mixture. More specifically, cross-linking the collagen mixtureincludes cross-linking the collagen (e.g., by forming a bond betweencollagen fibrils). In some embodiments, the cross-linking includespreparing a solution of glutaraldehyde and phosphate buffer. Forexample, in some embodiments, a solution of 25% glutaraldehyde solutionand 0.02 M phosphate buffer is prepared. Specifically, for example, asolution of 50 μL of the 25% glutaraldehyde solution and 100 mL 0.02 Mphosphate buffer with a pH of 7.4 is prepared. The collagen mixture isplaced into the solution for a desired length of time for cross-linkingto occur. For example, in some embodiments, the collagen mixture isplaced into the solution and is cross-linked for about thirty minutes atroom temperature. The cross-linked collagen mixture is removed from thesolution.

At step 184, the method 180 optionally includes disposing the collagenmixture into at least one mold having a desired shape. The disposing caninclude scooping or pouring the collagen mixture into the mold. Thecollagen mixture can be molded into any desired shape for repair of boneat a target repair site. For example, the mold can have a shape thatmimics or correlates to the shape of the target repair site. In anotherexample, the collagen mixture can be molded into the shape of a circle(e.g., FIG. 2A), polygon (e.g., FIG. 3), square (e.g., FIG. 4), oval(e.g., FIG. 5), star (e.g., FIG. 6), cone, cylinder (e.g., FIG. 7),rectangle (e.g., FIG. 8), cube, disk, dowel, plug (e.g., FIG. 9), orother suitable shape. More specifically, in some embodiments, thecollagen mixture is molded into a three-dimensional rectangular shapedimplant that has a length about three times greater than a width of theimplant. In some embodiments, the collagen mixture is molded into animplant that has a width up to about four times a depth of the implant.For example, the collagen mixture can be molded into an implant that isabout 50 mm in length, 15 mm in width, and 4 mm in depth. The disposingthe collagen mixture into a mold can include disposing the mixture intothe mold while the mixture is in a flowable form (e.g., in the form of aslurry or foam).

At step 186, the method 180 includes lyophilizing the collagen mixtureto form a porous bone graft material. In some embodiments, the method180 results in the bone graft material being about 15% to about 20% byweight collagen, about 55% to about 70% by weight bioactive glass, andabout 15% to about 30% by weight calcium phosphate. The bioactive glassand the calcium phosphate together can be about 80% to about 85% byweight of the resulting bone graft material. When the bone graftmaterial is lyophilized, the collagen can form a hardened, brittle, orotherwise dry cracker-like material. In some embodiments, the porousbone graft material has about 70% porosity. The bone graft material isneither compressive molded nor annealed.

In some embodiments, the method optionally includes wetting (i.e.,hydrating) the bone graft material. For example, the lyophilized bonegraft material can be wetted with a suitable solution, as describedabove. In some embodiments, the wetting includes wetting the bone graftmaterial with a suitable solution that includes bioactive glassparticles. In some embodiments, the method optionally includes disposingbioactive glass particles onto the wetted bone graft material, forexample by sprinkling the bioactive glass particles onto a surface ofthe wetted bone graft material. Upon wetting (or re-wetting), at least aportion of the bone graft material (e.g., the collagen body portion) canbe flexible and/or moldable.

The method optionally includes packaging the porous bone graft material.For example, the bone graft material can be placed in a foil wrapper andsealed therein.

The method optionally includes irradiating the porous bone graftmaterial. The irradiating can help kill any bacteria or othercontaminants that may be present in or on the bone graft material. Thebone graft material can be irradiated at any appropriate level forsterilizing the bone graft material. For example, the bone graftmaterial can be gamma irradiated at about 25-40 kGy. In someembodiments, the bone graft material is irradiated after the packagingof the material. Irradiation of the bone graft material in the foilpackaging, for example, can help ensure the bone graft material remainssterile during shipment of the bone graft material from the manufacturerto a patient treatment facility.

A bone graft procedure according to an embodiment includes a method forimplanting a bone graft material or composition (including any bonegraft material or composition described herein) at a target repair sitewithin a body of a patient. The bone graft procedure optionally includespreparing the target repair site of the bone or bony structure withinthe patient's body to receive the bone graft material. Preparation ofthe target repair site can include cleansing the site to remove foreignmaterials, loose bone fragments or powder, or other potentially harmfulmaterials. In some procedures, preparation of the target repair siteincludes re-shaping the site, for example, by removing a portion of theperimeter of the site so that the site has a desired shape.

The bone graft procedure includes selecting a bone graft material. Forexample, in some embodiments, a physician or other healthcare providercan select a bone graft material having a shape corresponding to a shapeof the target repair site. In other embodiments, a physician can selecta flowable or moldable bone graft material. For example, the physiciancan select a bone graft material configured to be manually molded (e.g.,by the physician).

The bone graft procedure optionally includes shaping the bone graftmaterial for placement at the target repair site. For example, thephysician can manually manipulate (e.g., squeeze, pinch, stretch, etc.)the bone graft material (e.g., when the bone graft material is in thesponge-like form). In another example, the physician can pour a flowablebone graft material into a mold and dry the material so that thematerial retains the shape of the mold, in a similar manner as describedwith respect to the method 180 of making a bone graft material above. Insome embodiments, shaping the bone graft material includes cutting thebone graft material into a desired shape.

The bone graft procedure optionally includes wetting the bone graftmaterial with a suitable solution. In some embodiments, the suitablesolution includes bioactive glass particles. In some embodiments,bioactive glass particles are disposed on the bone graft material afterthe material is wetted with the suitable solution.

The bone graft procedure includes positioning the bone graft material atthe target repair site. In some embodiments, positioning the bone graftmaterial includes injecting the bone graft material in a flowable stateinto the target repair site. For example, the bone graft material can bein the form of a slurry, foam, paste, solution, or the like, which isinjected into the target repair site via a syringe. In some embodiments,positioning the bone graft material includes placing a bone graftmaterial in a dried or sponge-like form into the target repair site. Forexample, a dried or sponge-like bone graft material having a shapecorresponding to the shape of the target repair site can be positionedso that the shape of the bone graft material is aligned with the shapeof the target repair site. In this manner, the material is suitable forthe repair of substantially any shaped target repair site.

Optionally, at the physician's discretion, the bone graft procedureincludes wetting the bone graft material with a suitable solution afterpositioning the bone graft material at the target repair site. In someembodiments, the bone graft material is wetted with a fluid from thepatient's body. For example, blood or plasma from the patient's body canbe disposed on or permitted to flow to the bone graft material.

The bone graft procedure optionally includes closing an aperture in thepatient's body that provided access to the target repair site. Forexample, a skin flap can be repositioned over the implanted bone graftmaterial. In some embodiments, sutures, staples, or another closuremechanism are used to help close the aperture in the patient's body. Thepatient can be monitored for symptoms of complication (e.g., infection,rejection of the bone graft material), as well as for regrowth of boneat the target repair site.

While various embodiments have been described herein, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and steps described above indicate certainevents occurring in certain order, those of ordinary skill in the arthaving the benefit of this disclosure would recognize that the orderingof certain steps may be modified and that such modifications are inaccordance with the variations of the invention. For example, in someembodiments, the collagen mixture can be cross-linked after beingdisposed in the mold. In another example, in some embodiments, thecollagen mixture can be lyophilized before, or both before and afterbeing cross-linked. In another example, in some embodiments, thematerial is irradiated prior to packaging the material. Furthermore,each activity is not required for making the bone graft material. Forexample, in some embodiments, a collagen need not be prepared asdescribed prior to mixing the collagen with calcium phosphate.Additionally, certain of the steps may be performed concurrently in aparallel process when possible, as well as performed sequentially asdescribed above. The embodiments have been particularly shown anddescribed, but it will be understood that various changes in form anddetails may be made.

For example, although the method for making a bone graft materialincludes lyophilizing the mixture of collagen and calcium phosphate(and, optionally, bioactive glass), in other embodiments, a mixture canbe dried in a different manner. For example, in some embodiments, amixture of collagen and calcium phosphate (and, optionally, bioactiveglass) can be heat dried. A dry-heat process can also be configured toconcurrently sterilize the mixture.

Although the bone graft compositions (or materials) have been describedherein as being in a certain form (e.g., flowable, dried, sponge-like),in some embodiments, a bone graft composition can have a firstconfiguration in which the composition is in a first form and a secondconfiguration in which the composition is in a second form differentthan the first form. For example, in some embodiments, a bone graftcomposition includes a collagen matrix in a first form (e.g., a driedform) for delivery from the manufacturing facility to the patienttreatment facility, and in a second form (e.g., the sponge-like form)for implantation at the target repair site of the patient's body.

Specific examples of bone graft compositions according to embodimentsare now described, with reference to the following cell preparation andsample preparation procedures.

Example 1 Cell Preparation

Osteoblast-like MG-63 cells were prepared per American Type CultureCollection (“ATCC”) instructions. From passage three, the MG63 cellswere stored frozen at −70° C. in Eagle's minimum essential medium(“EMEM”) with 20% fetal bovine serum (“FBS”). In preparation for use,the MG63 cells were thawed and then grown for at least three passagesusing EMEM. After three passages, the cells were transferred toDulbecco's modified EMEM (“DMEM”). The DMEM contained no glucose and wassupplemented with ascorbic acid (50 μg/mL), 10 mM β-glycerophosphate, 50UI/mL Penicillin-Streptomycin and 10% FBS. The cells were grown for atleast three more passages at these conditions. The cells were maintainedat 37±1° C. in a humidified incubator with 5±1% CO₂. The media waschanged every 2 to 3 days. After the cells became confluent on the lastpassage, they were harvested and counted.

Example 2 Sample Preparation

Test samples of bone graft compositions including collagen, bioactiveglass, and calcium phosphate (60% HA/40% TCP) and test samples of bonegraft compositions including collagen and bioactive glass were prepared.The following preparation and testing was performed for each bone graftcomposition test sample. Each compound (i.e., collagen, bioactive glass,and calcium phosphate, as applicable; ratios of compounds each testsample are described below in Examples 3 through 8) was weighedaseptically using an analytical scale under a biological safety cabinetand transferred to a sterile non-treated tissue culture Petri dish(60×15 mm). A 30 mg mixture of the compounds in the specified ratio wasaseptically prepared. The 30 mg mixture was transferred to and dividedamongst four Petri dishes, each non-treated. A 1 mL PBS was added toeach dish. Each dish was then vigorously swirled by hand on a desksurface. Each dish was also spun in a centrifuge at 3000 rpm for 5minutes for even distribution of the composition on the dishes and tostick the compounds to the dishes.

Osteoblast-like MG-63 cells prepared according to Example 1 werere-suspended in media with supplements to a seeding concentration ofapproximately 2×10⁴ cell/cm². Next, 5 mL of the cells with media wascarefully added to each of the four dishes with the bone graftcompositions. The dishes were each gently swirled to spread thecomposition and cells. The cells were incubated in the dishes at 37±1°C. in a humidified incubator with 5±1% CO₂.

A control with collagen and calcium phosphate, but no bioactive glass,was prepared according to the foregoing steps. The control included amixture of 10 mg collagen and 20 mg calcium phosphate. The 30 mgcollagen and calcium phosphate mixture was transferred to and dividedamongst four Petri dishes. Cells were added to each of the four dishesin the same manner described above.

Cells were observed daily for 11 days. The media was changed carefullyevery 3 days. Cell confluence was observed and recorded at days 5 and11. The percentage of cell confluence indicated a percentage coverage ofthe bottom of the Petri dish by the cultured cells at different phasesof growth. As used herein, aggregation refers to cells composed of denseclusters of separate units and splicing of cells. Any differences (e.g.,no cell growth, loose cells, and change in cell appearance) between thesamples, the cell culture control, and the collagen-calcium phosphatecontrol were recorded and pictures were taken of representative plates.

On day 11, methanol was added to fix the cells. The cells were staineddirectly in the dishes using 3 staining methods: Calcium Stain Kit(modified Van Kossa staining), Trichrome staining (modified Masson'sstaining), and H&E (Hemotoxylin & Eosin) staining. The staining methodsaided in differentiating cells and their metabolites, if present. Thedifferent staining methods also aided in observing changes in cellmorphology in the various bone graft formulations.

Consideration regarding mineralization of each bone graft formulationwas based on the Calcium staining. According to the staining kit notes,cells with accumulated dispersed calcium must stain in gray color versusblack staining for calcium in mass deposits. Mineralization level, as apercentage, was assumed by calculating a ratio based on the size of thecalcium crystals and counting of bioactive glass crystals in multiplefields around the dish surface with gray formations on the surface ofcrystals and around borders of them (e.g., aureole, fluffycollagen-cellular fringe, etc.) versus crystals which looked intact(e.g., clear borders, no changes). The Trichrome and H&E Staining wereperformed to more clearly visualize changes of collagen scaffolds andchanges in cell appearance and density. Generally, the brighter thecolor, the higher the density of cells.

Example 3 Collagen:Bioactive Glass:Calcium Phosphate (15:65:20)

A test sample of a bone graft composition including 15% collagen, 65%bioactive glass, and 20% calcium phosphate (60% HA/40% TCP) was preparedaccording to Example 2. Cell confluence was determined based on visualobservation of the sample without staining, as shown in the images ofthe sample taken on day 2, day 5, and day 11 in FIGS. 11A-11C,respectively. Cell confluence reached nearly an average of 93% by day 5.Cell confluence reached about 100% by day 11. No cytotoxicity wasobserved when compared with cell controls. A chart of cell confluence ofthis Example 3 compared to Examples 4 and 5 below is shown in FIG. 13.

Referring to FIG. 12A, visual observation under light microscopy of theVan Kossa stained cells showed pink, large, prolonged cells withdark-red big nuclei. The cells were in a fairly thick multilayer withmany aggregations. Some crystals and phosphates appeared intact, butwith grayish aureole. Some of the bioactive glass crystals andphosphates had collagen-cellular fringe and appeared very fluffy. Thismay be due to the presence of dispersed calcium accumulated by thecells. The collagen scaffolds show high cell growth. The compositionappeared to achieve approximately 60% bioactive glass mineralizationwith medium size calcium crystals. A chart of mineralization levels ofthis Example 3 compared to Examples 4 and 5 below is shown in FIG. 14.

Referring to FIG. 12B, visual observation under light microscopy of theH&E stained cells showed pink-purple cells of regular shape with bluebig nuclei. The cells were in a fairly dense multilayer surrounding mostof the bone graft composition. Many phosphates and bioactive glasscrystals were densely covered by cells or had layers of climbing cells.The collagen scaffolds were rich by cells.

Referring to FIG. 12C, visual observation under light microscopy of theTrichrome stained cells showed bright red cells of regular shape. Thecells were mostly in a dense multilayer, especially around differentscaffolds (collagen, bioactive glass crystals, calcium phosphates), withsome aggregations. Many (about 90%-95%) bioactive glass crystals andcalcium phosphates were densely covered by cells, some with layers ofclimbing cells or collagen-cellular fringe. Few bioactive glass crystalsappeared intact.

Example 4 Collagen:Bioactive Glass:Calcium Phosphate (15:60:25)

A test sample of a bone graft composition including 15% collagen, 60%bioactive glass, and 25% calcium phosphate (60% HA/40% TCP) was preparedaccording to Example 2. Cell confluence was determined based on visualobservation of the sample without staining, as shown in the images ofthe sample taken on day 2, day 5, and day 11 in FIGS. 15A-15C,respectively. Cell confluence reached nearly 75% by day 5. Cellconfluence reached about 97% in average at the end by day 11. Nocytotoxicity was observed when compared with cell controls. A chart ofcell confluence of this Example 4 compared to Examples 3 and 5 (below)is shown in FIG. 13.

Referring to FIG. 16A, visual observation under light microscopy of theVan Kossa stained cells showed pink, large prolonged regular shapedcells with big red nuclei. Cell growth was uneven, with some areashaving a monolayer or poor growth. Many crystals and phosphates hadlayers of climbing cells or were densely covered by cells. Many crystalsappeared fluffy. Many crystals were covered by grayish collagen-cellularfringe, which appeared to be dispersed calcium that could be consideredin the cells. Some crystal had clear borders surrounded by grayishaureole. The composition appeared to achieve approximately 77% bioactiveglass mineralization with large calcium crystals. A chart ofmineralization levels of this Example 4 compared to Examples 3 and 5(below) is shown in FIG. 14.

Referring to FIG. 16B, visual observation under light microscopy of theH&E stained cells showed purple-pink, large, regular shaped cells withblue big nuclei. The cells were mostly in thick, dense multilayer. Someareas showed a thin layer of cells. Much of the bone graft compositionwas covered by a dense net of cells.

Referring to FIG. 16C, visual observation under light microscopy of theTrichrome stained cells showed red, normal cells in a very densemultilayer. Some phosphates granules were surrounded by a very thinlayer of cells, especially on the borders of the dishes. Most (about95%) of the bone graft composition was densely covered by cells.

Example 5 Collagen:Bioactive Glass:Calcium Phosphate (15:55:30)

A test sample of a bone graft composition including 15% collagen, 55%bioactive glass, and 30% calcium phosphate (60% HA/40% TCP) was preparedaccording to Example 2. Cell confluence was determined based on visualobservation of the sample without staining, as shown in the images ofthe sample taken on day 2, day 5, and day 11 in FIGS. 17A-17C,respectively. Cell confluence reached nearly 80% by day 5. Cellconfluence reached about 93% by day 11. No cytotoxicity was observedwhen compared with cell controls. A chart of cell confluence of thisExample 5 compared to Examples 3 and 4 above is shown in FIG. 13.

Referring to FIG. 18A, visual observation under light microscopy of theVan Kossa stained cells showed large prolonged regular shaped cells withbig nuclei in fairly thick high density multilayer especially around thebone graft composition. Many phosphate granules and bioactive glasscrystals were observed to have grayish aureole and fuzzy borders, somewith layers of climbing cells. Some of the bioactive glass crystals havecollagen-cellular fringe on the surface and appear fluffy and grayish.This appeared to be due to the presence of dispersed calcium accumulatedby cells, as other bioactive glass crystals have mostly clear bordersand appear intact. The composition appeared to achieve approximately 60%bioactive glass mineralization with medium size crystals. A chart ofmineralization levels of this Example 5 compared to Examples 3 and 4above is shown in FIG. 14.

Referring to FIG. 18B, visual observation under light microscopy of theH&E stained cells showed that the cells appeared healthy and regular inshape with large nuclei. The cells were mostly in fairly thick,high-density multilayer with some aggregations. Many crystals werecovered by the cells net, some with attached collagen fringe with highcells growth. Many crystals and phosphate had layers of climbing cellson the edges.

Referring to FIG. 18C, visual observation under light microscopy of theTrichrome stained cells showed many phosphate granules and bioactiveglass crystals were covered by a net of dense cells. The cells appearednormal and of regular shape in a thick multilayer. Some crystals hadcellular-collagen fringe. The collagen scaffolds had high cell growth,but some of the bone graft composition remained intact.

Each of the bone graft compositions in Examples 3, 4, and 5 promotedgood cell proliferation and had good cytocompatibility. Cells appearedhealthy, mostly in multilayer of different density depending on thecomposition. Cells were large, prolonged or star-like, and regularlyshaped with big nuclei, which is typical growth for osteoblast cells.

Example 6 Collagen:Bioactive Glass (20:80)

A test sample of a bone graft composition including 20% collagen and 80%bioactive glass was prepared according to Example 2. Cell confluence wasdetermined based on visual observation of the sample without staining,as shown in the images in FIGS. 21A-21C of the sample taken on day 2,day 5, and day 11, respectively. Cell confluence reached 90% by day 5.Cell confluence reached 100% by day 11. No cytotoxicity was observedwhen compared with cell controls. A chart of cell confluence of thisExample 6 compared to Examples 7 and 8 below is shown in FIG. 19.

Referring to FIG. 22A, visual observation under light microscopy of theVan Kossa stained cells showed prolonged cells with pink cytoplasm andlarge red nuclei. Also observed were black bioactive glass crystals.Many crystals showed grayish fuzzy borders, some with fluffy aureole,which appeared to be high cell density with dispersed calcium. Thecomposition appeared to achieve approximately 20% bioactive glassmineralization. A chart of mineralization levels of this Example 6compared to Examples 7 and 8 below is shown in FIG. 20.

Referring to FIG. 22B, visual observation under light microscopy of theH&E stained cells showed regular shaped, purple-pink cells with largenuclei. Also observed were many crystals with cells on their surface.Most of the crystals appeared intact.

Referring to FIG. 22C, visual observation under light microscopy of theTrichrome stained cells showed bright red, regular cells with black-bluelarge nuclei in a thick multilayer. Many crystals were observed withattached collagen with high cell growth, and with aureole or bright redcollagen because of cells growing through. Some crystals were withoutcells and were intact. Also observed were some chunks of dark-bluecollagen.

Example 7 Collagen:Bioactive Glass (40:60)

A test sample of a bone graft composition including 40% collagen and 60%bioactive glass was prepared according to Example 2. Cell confluence wasdetermined based on visual observation of the sample without staining,as shown in the images in FIGS. 23A-23C of the sample taken on day 2,day 5, and day 11, respectively. Cell confluence reached 90% by day 5.Cell confluence reached 100% by day 11. No cytotoxicity was observedwhen compared with cell controls. A chart of cell confluence of thisExample 7 compared to Examples 6 and 8 (below) is shown in FIG. 19.

Referring to FIG. 24A, visual observation under light microscopy of theVan Kossa stained cells showed regular shaped pink cells with large rednuclei. Also observed were black bioactive glass crystals. Some crystalsshowed fuzzy borders and fluffy grayish aureole around the borders,which appeared to be covered by cells with dispersed calcium. Thecomposition appeared to achieve approximately 73% bioactive glassmineralization with large calcium crystals. A chart of mineralizationlevels of this Example 7 compared to Examples 6 and 8 (below) is shownin FIG. 20.

Referring to FIG. 24B, visual observation under light microscopy of theH&E stained cells showed regular shaped, purple-pink cells with largeblue nuclei. The bioactive glass crystals appeared to mostly be intact.Some crystals had cells growing on their surface.

Referring to FIGS. 24C and 24D, visual observation under lightmicroscopy of the Trichrome stained cells showed multilayer of healthyregular shaped cells with large black nuclei. Crystals were mostlyintact. Some crystals had attached collagen fringe with high cellgrowth. Some crystals were covered just by cells without collagen.Structure resembling bone formation was clearly observed.

Example 8 Collagen:Bioactive Glass (60:40)

A test sample of a bone graft composition including 60% collagen and 40%bioactive glass was prepared according to Example 2. Cell confluence wasdetermined based on visual observation of the sample without staining,as shown in the images in FIGS. 25A-25C of the sample taken on day 2,day 5, and day 11, respectively. Cell confluence reached 70% by day 5.Cell confluence reached 95% by day 11. No cytotoxicity was observed whencompared with cell controls. A chart of cell confluence of this Example8 compared to Examples 6 and 7 above is shown in FIG. 19.

Referring to FIG. 26A, visual observation under light microscopy of theVan Kossa stained cells showed regular shaped pink cells with large rednuclei. Also observed were black bioactive glass crystals, many withstuck collagen fibers. Some crystals had fuzzy borders and fluffygrayish aureole around the borders, which might be covered by cells withdispersed calcium. The composition appeared to achieve approximately 67%bioactive glass mineralization with smaller calcium crystals. A chart ofmineralization levels of this Example 8 compared to Examples 6 and 7above is shown in FIG. 20.

Referring to FIG. 26B, visual observation under light microscopy of theH&E stained cells showed regular shaped, purple-pink cells with largeblue nuclei. The crystals appeared mostly intact. Some crystals wereobserved with cells on their surface.

Referring to FIG. 26C, visual observation under light microscopy of theTrichrome stained cells showed multilayer of healthy regular shapedcells with large black nuclei. The crystals were mostly intact. Somecrystals had attached collagen fringe with high cell growth. Somecrystals were covered just by cells without collagen.

Each of the bone graft compositions in Examples 6, 7, and 8 promotedgood cell proliferation, had no cytotoxicity, and had goodcytocompatibility. Cells were large, prolonged or star-like, andregularly shaped with big nuclei, which is typical growth for osteoblastcells.

CONCLUSION

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having any combination or sub-combination of any featuresand/or components from any of the embodiments described herein. Thespecific configurations of the various components can also be varied.For example, the size and specific shape of the various components canbe different than the embodiments shown, while still providing thefunctions as described herein.

Thus, the breadth and scope of the invention should not be limited byany of the above-described embodiments, but should be defined only inaccordance with the following claims and their equivalents. The previousdescription of the embodiments is provided to enable any person skilledin the art to make or use the invention. While the invention has beenparticularly shown and described with reference to embodiments thereof,it will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention.

What is claimed is:
 1. A bone graft composition comprising: about 15% toabout 20% by weight collagen; about 55% to about 70% by weight bioactiveglass, the bioactive glass including particles within a range of about53 μm to about 425 μm in size, at least 85% of the bioactive glassparticles being within a range of 212 μm to 425 μm in size; and about15% to about 30% by weight calcium phosphate.
 2. The bone graftcomposition of claim 1, wherein the bioactive glass particles and thecalcium phosphate together are about 80% to about 85% by weight of thebone graft composition.
 3. The bone graft composition of claim 1,wherein the collagen is type I collagen.
 4. The bone graft compositionof claim 1, wherein the bioactive glass includes particles having asmooth surface.
 5. The bone graft composition of claim 1, wherein thecalcium phosphate comprises about 40% to about 80% tricalcium phosphateand about 20% to about 60% hydroxyapatite.
 6. The bone graft compositionof claim 5, further comprising about 15% by weight type I collagen,about 55% to about 65% by weight bioactive glass, and about 20% to about30% by weight calcium phosphate.
 7. The bone graft composition of claim6, wherein a weight ratio of the collagen to the bioactive glass to thecalcium phosphate is about 15%:55%:30%, respectively.
 8. The bone graftcomposition of claim 6, wherein a weight ratio of the collagen to thebioactive glass to the calcium phosphate is about 15%:60%:25%,respectively.
 9. The bone graft composition of claim 6, wherein a weightratio of the collagen to the bioactive glass to the calcium phosphate isabout 15%:65%:20%, respectively.
 10. The bone graft composition of claim1, wherein the bioactive glass is 45S5 glass.
 11. A bone graftcomposition, comprising: a collagen carrier; and a bioactive glassdispersed throughout the collagen carrier, the bioactive glass includingparticles having a size within a range of about 53 μm to about 425 μm,at least 85% of the bioactive glass particles having a size within arange of 212 μm to 425 μm; wherein the collagen carrier is about 20% toabout 60% by weight of the bone graft composition and the bioactiveglass is about 40% to about 80% by weight of the bone graft composition.12. The bone graft composition of claim 11, wherein the collagen carrieris porous.
 13. The bone graft composition of claim 11, wherein thebioactive glass has a smooth surface.
 14. The bone graft composition ofclaim 11, wherein the bioactive glass is about 80% by weight of the bonegraft composition.
 15. The bone graft composition of claim 11, whereinthe bioactive glass is about 60% by weight of the bone graftcomposition.
 16. The bone graft composition of claim 11, wherein thebioactive glass is 45S5 glass.
 17. The bone graft composition of claim11, wherein the collagen carrier includes type I collagen.
 18. The bonegraft composition of claim 11, wherein the collagen carrier and thebioactive glass collectively comprise 100% by weight of the bone graftcomposition.
 19. A bone graft composition, comprising: a collagencarrier comprising about 10% to about 20% by weight of the bone graftcomposition; a plurality of bioactive glass particles within the rangeof about 53 μm to about 425 μm in size and dispersed throughout thecollagen carrier, at least 85% of the plurality of bioactive glassparticles being within the range of 212 μm to 425 μm in size, theplurality of bioactive glass particles comprising about 25% to about 80%by weight of the bone graft composition, the plurality of bioactiveglass particles each having a smooth surface.
 20. The bone graftcomposition of claim 19, wherein the collagen includes type I collagen.